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Abstract:

The present invention discloses a method of improving the fermentation
yield of polyunsaturated fatty acids, in which Schizochytrium limacinum
is used as production strain to produce polyunsaturated fatty acids
(PUFA), and glycine betaine or trehalose is added to the fermentation
medium. In the present invention, after Schizochytrium limacinum
fermentation system is treated with exogenous glycine betaine, the yield
of PUFA produced by fermentation with Schizochytrium limacinum can be
greatly improved. The present invention significantly improves the yield
of PUFA produced by microorganism, reduces the cost without harming the
environment, and saves manpower and material resources by simple and
effective regulation of fermentation, and thus the method is simple,
convenient and cost-effective.

Claims:

1. A method for improving the production of polyunsaturated fatty acid,
in which Schizochytrium limacinum utilizes fermentation medium to produce
the polyunsaturated fatty acid, the fermentation medium containing a
compatible solute.

2. The method for improving the production of polyunsaturated fatty acid
according to claim 1, wherein said compatible solute is selected for a
group consisting of glycine betaine and trehalose.

3. The method for improving the production of polyunsaturated fatty acid
according to claim 1, wherein said Schizochytrium limacinum is
Schizochytrium limacinum HX-308 (CCTCC No. M209059).

4. The method for improving the production of polyunsaturated fatty acid
according to claim 2, wherein in the fermentation medium, the
concentration of the glycine betaine is from 10 to 100 mmol/L.

5. The method for improving the production of polyunsaturated fatty acid
according to claim 4, wherein in the fermentation medium, the
concentration of the glycine betaine is from 10 to 70 mmol/L.

6. The method for improving the production of polyunsaturated fatty acid
according to claim 5, wherein in the fermentation medium, the
concentration of the glycine betaine is 40 mmol/L.

7. The method for improving the production of polyunsaturated fatty acid
according to claim 2, wherein in the fermentation medium, the
concentration of the trehalose is from 10 to 200 mmol/L.

8. The method for improving the production of polyunsaturated fatty acid
according to claim 7, wherein in the fermentation medium, the
concentration of the trehalose is from 40 to 200 mmol/L.

9. The method of for improving the production of polyunsaturated fatty
acid according to claim 8, wherein in the fermentation medium, the
concentration of the trehalose is 80 mmol/L.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a method for enhancing the
fermentation yield of polyunsaturated fatty acids, belonging to the field
of biotechnology.

BACKGROUND ART

[0002] Polyunsaturated fatty acids (PUFA), which are important constituent
of biofilm of the cells and organisms, can regulate cell configuration,
homeostasis, phase transition, and the permeability of the cell membrane,
and also adjust the membrane-related physiological processes, so they can
influence the chemical composition of the cell, the signal transmission,
immunity and cold adaptation, as well as the occurrence of diseases
associated therewith. PUFA can be converted into metabolites adjusting
some physiological function of human. Docosahexaenoic acid (DHA), which
is the main component of PUFA in cell membranes, has important
physiological functions, such as enhancing memory enhancement, improving
intelligence, lowering blood lipids, regulating the immune system and
other effects, but also can prevent and treat cardiovascular disease ,
such as cancer, etc. Docosapentaenoic acid (DPA), which is a long-chain
unsaturated fatty acid in the human colostrum only, is the major
constituent of the human brain tissue and nerve cells and is essential to
the development of the nervous system and vision, the formation of the
brain, and enhancement of memory of the infant. Furthermore, DPA can
promote and improve the body's immunity. DPA and DHA have a synergistic
effect and show a greater therapeutic effect on type II diabetes,
rheumatoid arthritis, psoriasis, asthma, ulcerative colitis and
enteritis, etc., and thus have a great commercial value. In recent years,
scientists have carried out a study of fermentation production of DHA
with marine microorganisms. The common microorganisms include
Crypthecodinium cohnii and Thraustochytrium spp., etc.Compatible solutes,
which are cell metabolic intermediates, non-toxic, can adjust the osmotic
pressure and prevent the radical change of the ion concentration in the
cells. When the intracellular osmolality changes radically, e.g. when
external osmotic pressure is elevated, the cells begin to produce or
absorb several small solutes, such as trehalose, betaine and certain
amino acids in order to improve the water activity within the cell to
maintain the balance of osmotic pressure inside and outside of the cell,
and meanwhile prevent the outflow of the cell moisture and salinity
intrusion. These small solutes are "compatible solute".

[0003] Scientists have carried out a study of fermentation production of
DHA with marine microorganisms. In summary, patents published in China
include the following four aspects: 1, regarding mutation screening
methods of DHA-producing strain, such as "industrial application of
marine fungi Schizochytrium limacinum OUC88" (200410075426.X), Ocean
University of China; "a docosahexaenoic acid-producing strain and
mutation screening methods and their application" (200910033493.8),
Nanjing University of Technology; 2, regarding the composition of the
medium, such as "a Schizochytrium limacinum and preparation of DHA oil
using the same" (CN200910033869.5), Nanjing University of Technology,
etc.; 3, regarding the extraction and purification of fats, such as
"extraction and purification of DHA-rich fatty acid from Crypthecodinium
cohnii" (200710025079.3), Nanjing University of Technology; "method of
extracting unsaturated fatty acid DHA from the fermentation broth of
Dinoflagellates" (CN200910159368.1), etc. Inner Mongolia Kingdomway
Pharmaceutical Co., Ltd., etc.; 4, regarding DHA application, such as
"maternal nutritional food (CN200610000658.8), Zhu Yan Hong etc.,
"preparation of ready-to-eat fish ball slice" (CN200510045178.9), Chen
Yi. Currently, method of increasing the content of fatty acids by simple
regulation of the fermentation has not been reported yet.

[0004] Patents concerning compatible solutes published in China generally
include the following two aspects: 1, regarding extraction and
preparation of compatible solutes, such as "preparation and use of an
organic green feed additive" (CN200910109142.0), Shen Guangrong;
"preparation of betaine" (CN00811384.X), AkzoNobel; "new method of
detection and extraction of compatible solutes ectoine from neutral
halophilic bacterium Halomonas salina" (CN200610135272.8), etc., Third
Institute of Oceanography of the State Oceanic Administration; 2,
regarding the application of compatible solutes. More is applied to
improve the growth performance of the yield of animals and plants, etc.,
such as "a composite preparation for promoting the growth of aquatic
animals to improve the meat quality and its preparation method"
(CN200910307231.6), Tianjin Shengji Group Co., Ltd.; "method of improving
crop yields" (95197919.1, 95197917.5 etc.), Cart limited. Etc.; one is
just for microbial fermentation aspects: "a new process to improve the
fermentation yield of L-glutamic acid (CN200910067618. 9), Tianjin
University of Science and Technology.

[0006] The technical problems to be solved by the present invention is to
provide a simple and efficient method for increasing fermentation yield
of polyunsaturated fatty acids and reducing costs without harming the
environment and increasing the manpower and material resources.

[0007] To solve the above technical problem, the technical solution
adopted by the present invention is provided as follows:

[0008] A method of enhancing the fermentation yield of polyunsaturated
fatty acids, in which Schizochytrium limacinum is used as production
strain to yield polyunsaturated fatty acids, and compatible solute is
added to the fermentation medium.

[0009] Wherein said compatible solute is glycine betaine or trehalose.

[0010] In the fermentation medium, the concentration of the glycine
betaine is from 10 to 100 mmol/L, preferably from 10 to 70 mmol/L, and
most preferably 40 mmol/L.

[0011] In the fermentation medium, the concentration of the trehalose is
from 10 to 200 mmol/L, preferably from 40 to 200 mmol/L, preferably 80
mmol/L.

[0012] The yield of PUFA produced by fermentation with marine
microorganism can be improved by adding a small amount of exogenous
compatible solutes to the fermentation medium. Glycine betaine is the
main compatible solute which is generated when Schizochytrium limacinum
responds to the ambient pressure, and it is relatively cheap, about 40
rmb/kg. Trehalose is a compatible solute which is generated when the
microorganism responds to ambient pressure, about 70 rmb/kg.

[0013] Under fermentation conditions, to the medium producing DHA by
fermentation with Schizochytrium limacinum is added glycine betaine or
trehalose to enhance the yield of PUFA. When cultured marine
microorganisms are subjected to adverse fermentation conditions,
exogenous compatible solute is effective. External pressure factors
include high temperature, high-pressure, high-salt, hypertonia,
hypotonicity and drying, etc. Schizochytrium limacinum treated with
exogenous compatible solute can better cope with the changing environment
of the outside world and improve the yield of DHA. The compatible solute
is the stable substance in microbial cells, hence the beneficial effect
of the compatible solute is long-lasting.

[0014] Beneficial effects: in the present invention, after Schizochytrium
limacinum fermentation system is treated with exogenous glycine betaine,
yield of PUFA produced by fermentation with Schizochytrium limacinum can
be greatly improved, mass percentage of DPA in total fatty acids is
increased from 11.9% to 16.2%; mass percentage of DHA in total fatty
acids is increased from 44.1% to 49.8%; mass percentage of squalene is
increased from 0.8% to 1.7%; and mass percentage of saturated fatty acid
C14: 0 and C16: 0 in total fatty acid is significantly reduced,
respectively, from 10.0% to 5.1%, and from 24.4% to 20%. DHA yield is
increased from 3.9 g/L to 5.0 g/L, increased by 28%; the ratio of DHA in
the total biomass (mg/g) is increased from 57 to 72; total fatty acid
yield is increased from 8.8 g/L to 10 g/L .

[0015] After using appropriate amount of trehalose, DHA yield is increased
from 3.9 g/L to 7.5 g/L, increased by 92%; biomass is increased from 60
g/L to 76 g/L; the ratio of DHA in total biomass (mg/g) is increased from
57 to 99; yield of total fatty acids is increased from 8.8 g/L to 16.7
g/L

[0016] The present invention significantly improves the yield of PUFA
produced by microorganism and reduces cost without harming the
environment and increasing manpower and material resources by simple and
effective regulation of fermentation, and thus is simple, convenient and
cost-effective.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

[0017] According to the following examples, the present invention can be
better understood. The person skilled in the art, however, can easily
understand that the ratio of raw materials, process conditions and the
results are intended to illustrate the present invention only, and should
not and does not limit the invention specifically described in the
claims.

[0018] The detection method of the following Examples is the same as that
in the application titled "Schizochytrium limacinum and method of
production of DHA oil using the same" (Application No. 200910033869.5)

EXAMPLE 1

[0019] The strain is Schizochytrium limacinum HX-308 with deposit number
CCTCC No. M209059.

[0022] Cultural method: strain was inoculated into seed medium with
inoculation amount of 5% (v/v), cultured in a 500 mL shake flask for 24 h
to log phase under 170 r at 25° C., and inoculated into the
fermentation medium with addition of a certain amount of glycine betaine
(such as 10 mmol/L, 40 mmol/L, 70 mmol/L, 100 mmol/L) in inoculation
amount of 9% (v/v) for culturing; fermentation ceased when glucose
reached an amount of 0 g/L. The results are shown in Table 1.

[0026] Cultural method: the strain was inoculated into seed medium with
inoculation amount of 5% (v/v), cultured in a 500 mL shake flask for 24 h
to log phase under 170 r at 25° C., and inoculated into the
fermentation medium with addition of a certain amount of trehalose (such
as 10 mmol/L, 40 mmol/L, 80 mmol/L) in inoculation amount of 9% (v/v);
fermentation ceased when glucose reached an amount of 0 g/L. The results
are shown in Table 2.